Method of preparing protein standardized reagents



Nov. 13, 1956 T. E. WEICHSELBAUM 2,770,602

METHOD OF PREPARING PROTEIN STANDARDIZED REAGENTS Original Filed June 251951 FIGJ INVENTOR.

M U A B L E S H m E W E E R O D O E H T BY FIG.3

ATTORNEY Unite St IVIETHOD OF PREPARING PROTEIN STANDARDIZED REAGENTSTheodore E. Weichselbaum, Normandy, Mo., assignor to A. S. Aloe Company,St. Louis, Mo., a corporation of Missouri 4 Claims. (Cl. 252408) Thisinvention relates to certain new and useful improvements in methods ofpreparing quantitative laboratory standards of the type used in clinicalchemistry for verifying and correcting instrument calibration tables andgraphs. This application is a division of my co-pending applicationSerial No. 233,465, filed June 25, 1951, which in turn was acontinuation-in-part of application Serial No. 778,681, filed October 8,1947, and abandoned.

At the present time, in the performance of many routine analyses,clinical laboratories make use of physical colorimetry andspectrophotometry as well as the practice of various related technics,such as those requiring the employment of photoelectric instruments andthose involving titrimetry. Analyses of this character have exceedinglybroad application and in the physiological field, for diagnosticpurposes, are extensively used in the analysis of body fluids such asblood serum and plasma, urine, and spinal fluid. Such procedures,although routine or research in nature, normally require painstaking andlaborious preliminary steps, including the exacting preparation ofquantitative standards for color comparison purposes and for checkingthe accuracy of the instruments utilized so that any deviations in theoperation thereof will be precisely adjusted for by calibration.Customarily, whatever excess may remain of these standards after theaccomplishment of the color comparison or the instrument verification orcorrection procedure will usually be discarded since same are subject todeterioration, inadvertent dilution or contamination and, thus, theirfurther use at a later date will be productive of inaccuracy.Consequently, it is necessary to prepare such standards immediatelyprior to their use. This procedure is manifestly inherently uneconomicaland, furthermore, demands an element of proficiency on the part of theoperator in preparing the standard since any errors in the formationthereof will perforce cause the determination of false results.

Furthermore, the preparation of many of such standards are mostintricate and thereby demand marked skill and wide experience on thepart of the technician. It is evident that the amount of time spent byskilled personnel in preparing such reagents constitutes a severe lackof economy of such individuals time since it diverts them from theirimmediate research pursuits and thereby causes devotion of aconsiderable proportion of their time or merely ancillary preparatorysteps. With the developing shortage of highly trained scientificpersonnel, it is readily recognized that their efforts should at alltimes be directed solely to the problem at hand and not dissipated onpreparatory procedures.

Therefore, it is a primary object of the present invention to provide amethod of preparing quantitative laboratory standards incorporating thepreparation of a dried, solid reagent of predetermined weight which maybe dissolved in a selected diluent to form a standard of predeterminedconcentration suitable for accurate quantitative measurement.

tes atet It is a further object of the present invention to provide amethod for preparing quantitative laboratory standards which comprisesthe desiccation of a predetermined quantity of a solution of knownconcentration and the maintenance of such desiccated portion underconditions wherein it will retain its properties indefinitely.

It is an additional object of the present invention to provide a methodfor preparing quantitative laboratory standards which may be accuratelyand reliably performed by relatively unskilled operators.

It is a still further object of the present invention to provide amethod of preparing quantitative laboratory standards which is highlyeconomical, simple in performance, and convenient.

With the above and other objects in view, my invention resides in thenovel methods and processes presently described and pointed out in theclaims.

In the accompanying drawing Figure l is a side elevational view of apreferred form of reagent ampule for use in the performance of themethod of the present invention;

Figure 2 is a vertical cross-sectional view of the ampule shown inFigure 2; and

Figure 3 is a horizontal sectional view taken along line 33 of Figure l.

The method herein comprises the formation of a solution of desireconcentration; the measuring of a predetermined quantity of the preparedsolution and the depositing of the same within an ampule or otherenclosable container; the dehydration or desiccation of the measuredquantity in the ampule by any suitable means, such as by evaporation orby lyophilization, as in the manner set forth in United States LettersPatent No. 2,225,774, issued to E. W. Flosdorf, December 24, 1940; thesealing of the ampule under vacuum; and the ultimate utilization of thewholly dried, solid reagent by rupture of the ampule and dissolution ofthe dried reagent in a predetermined quantity of selected diluent toreconstitute a quantitative standard of known concentration.

Referring now in more detail, and by reference characters to thedrawing, which illustrates a practical type of ampule for use in themethod herein taught, but is not a part of the present invention, Adesignates a blown or drawn glass ampule of essentially tubular form andintegrally comprising a lower or body portion 1, an intermediate orconstricted portion 2, and an upper portion 3 preferably ofsubstantially the same diametral size as the body portion 1, tapering atits upper end into an axially upwardly projecting sealing neck 4, thelatter being initially open for filling purposes and, subsequent tofilling, being sealed, as at 5, in a hot flame, such as a Bunsen burneror blow lamp. The constricted portion 2 is provided, midway of itslength, with a line of weakness or scratch line 6, and a small outwardlyblown or drawn nipple-like protuberance 7, the apex or point of which iscoincident with the scratch line 6.

In the performance of the method of this invention, the reagent ampule Ais filled with an accurately measured or weighed quantity of reagenthaving a precisely determined concentration suitable for making areagent solution of definite strength or normality when added to astated quantity of distilled water or other diluent. The quantity ofreagent thus deposited is dehydrated for reduction to a completelydried, solid state. This may be accomplished by lyophilization whereinthe sealing neck 4 which is, as yet, still open, is connected bysuitable rubber tubing to a high vacuum system (not shown), which may beof any conventional design or construction and the contents of theampule desiccated by freezing the liquid contents and conductingdesiccation by sublimation. This method is particularly advantageous indealing with various types of biological reagents which must be handledvery carefully to avoid impairment of their action. After the producthas been completely desiccated, the sealing neck 4 is sealed off and theproduct is completed, ready for labeling and final use. The preciseconditions of such dehydration methods depend upon the character of theparticular standard being prepared as some will require low temperaturedesiccation whereas others are reduced to a dry state by hightemperature vacuum procedures.

In some cases, such as with a bilirubin standard, lyophilization is notused but the dehydration process is carried out by evaporation procedurewith sealing of the desiccated material in an inert atmosphere, as willpresently be more fully described. The manner of desicca tion is thusdependent upon the particular substances involved.

Reagents so ampulized in wholly desiccated state will keep forindefinite periods without deterioration, remaining stable as to theiroriginal properties, so that an operator may use same, as will be shownbelow, whenever the particular occasion arises without fear of anyinaccuracy resulting through untoward impairment of the reagent.

When the operator desires to make a quantitative laboratory standard forclinical analytical purposes, the ampule A is manually broken along thescratch line 6 into two sections, and the contents washed by a smallquantity of the indicated diluent into the container in which suchstandard is to be made. It is to be particularly noted that exactquantitative transfer of the ampule contents must be made. Therefore,the desiccated material in both of the sections of the now broken ampulemust be completely dissolved and transferred. The rupturing of theampule A along the scratch line 6 will result in the formation of apouring lip to facilitate transference of the dissolved reagents intothe container in which the standard is being made. Successive washingsfrom a conventional laboratory wash bottle, or the like may be done toassure that all desiccated material has been totally dissolved andtransferred. The solution is then made up to the predetermined or statedvolume.

The quantitative standards so prepared by the present invention areparticularly fitted for utilization in verifying the calibration tablesand graphs of photoelectric colorimeters and spectrophotometers, as wellas providing color standards for visual colorimetry and for titrimetrictechnics. As illustrative of the method herein described, below arepresented two specific examples of the'preparation of a quantitativestandard for checking the accuracy of the type of calibration tablesupplied with a pre-calibrated spectrophotometer or of atransmittanceconcentration graph.

Example I The preparation of a hematin standard for use in the alkalinehematin technic for determining total hemoglobin is readily accomplishedby this method. Although hematin standards of various concentrationsmay, of course, be prepared, the example herewith relates to onerepresenting 18.7 grams percent hemoglobin equivalent. A one litersolution is formed by dissolving 2.776 grams of hematin in a .2 molarborate buffer, pH 9.4. After incubation and aging of the reagent thusmade, cc. thereof, containing a proportionate amount of hematin, namely13.88 mg., is deposited 'into the ampule A through the now open sealingneck 4. The 5 cc. of reagent is then subjected to lyophilization whichcomprises shell freezing by immersion of the ampule A into a suitablebath, such as isopropyl alcohol at 40 C. Subsequent to the freezingstep, and before thawing can occur, a highvacuum is applied in order toevacuate all water thereby leaving the reagent in a completelydesiccated, solid form. The ampule A is then sealed under vacuum.

To reconstitute the reagent for analytical use, the ampule A is brokenalong the line of weakness 6, and small amounts of distilled water, inthe neighborhood of 10 ml., are deposited in the upper and lowerportions 1,

verification of photoelectric and 3, of the ampule A for dissolving thesolid reagent. Thereon the portions 1, 3, are emptied into a 250 cc.flask. However, additional flushings of the ampule A may be made toassure quantitative dissolution of the entire reagent. The flask is thenmake up to the mark with the diluent to constitute a standard ofaccurate known strength, suitable for use as a standard in visualcolorimetry and for use as a standard for calibration ofspectrophotometric curves or calibration tables. As a verificationstandard, it has a transmittance equivalent to 18.7 grams percenthemoglobin.

Example II To prepare protein standards for use in determining the totalprotein and albumin content of blood, plasma is extracted from wholebeef blood and the protein content thereof discovered by conventionaltechnics. The beef plasma is then diluted with a saline solution,namely, .85 percent sodium chloride, in requiste amount to provide 360mg. of protein per 5 cc. Then 5 cc. of the reagent thus formed isdeposited in the ampule A and subjected to shell freezing anddesiccation, after which the ampule A issealed under vacuum. The solidreagent within the ampule A represents 9 grams percent total serumprotein equivalent. the ampule A is broken and flushed repeatedly withsmall amounts of the diluent, which in this case is 30 percenturea-thymol reagent, until the reagent is completely quantitativelydissolved, with transference being made to a ml. volumetric flask. Thediluent is then added to the flask to make up to the mark. Variousconcentrations of protein standard reagents may be made by this method,such as, one containing 280 mg. per 100 ml. which represents 7 gramspercent total protein material or one of the concentration of mg. per100 ml., which represents 4 grams percent total serum proteinequivalent, and being suitable for use in colorimetry and forcalibration for verification of photoelectric and spectrophotometriccurves or calibration tables.

Thus, by standards of the type above set forth, a technician mayaccordingly verify or correct calibration tables to assure accuratequantitative measurement.

The preparation of a bilirubin standard for use in quantitativelydetermining the presence of this pile pigment in serum or plasma, willserve as an example of the preparation of a reagent by evaporation. Inthis instance, a solution is made by dissolving 20 mg. bilirubin in 100cc. of chloroform. Then 5 cc. of the reagent thus formed is deposited inthe ampule A and subjected to evaporation to provide 1 mg. of bilirubinin a dry, solid state, after which the ampule A is sealed in an inertatmosphere, with nitrogen being the gas used. Thus, the air in theampule is replaced by nitrogen, the presence of which will preventoxidation of the bilirubin to biliverdin. The reagent is reconstitutedfor chemical and clinical analytical use by breaking the ampule A anddissolving completely the desiccated bilirubin with small amounts ofchloroform, and then quantitatively transferring the dissolved bilirubinby methyl alcohol to a 100 cc. volumetric flask. Methyl alcohol is thenadded to the flask to make up to the mark. As described hereinabove, itis understood that both sections of the broken ampule may be repeatedlywashed to assure exact and precise dissolution of all bilirubin. Thetotal amount of diluent utilized is roughly 95 percent methyl alcoholand 5 percent by volume chloroform; The solution so formed represents 1mg. percent bilirubin equivalent and will accordingly provide accuratelight transmittance for such concentration to serve as a reliableworking standard.

It should be understood that the present invention resides in the uniquemethod of providing dry, solid reagents of known strength from asolution of predetermined concentration, which reagent is adapted fiorreconstituting a solution of the identical concentration at a subsequentTo reconstitute the reagent,

time for quantitative measurement purposes in chemical and clinicalanalysis.

It is, of course, apparent that the present method is equally designedfor use by physicians, analytical chemists, and laboratory techniciansin both the medical and industrial fields, and obviously does notrequire, developed skill on the part of the operator. Thus, relativelyuntrained assistants may perform the method of the present inventionwithout substantial hazard of preparing an incorrect standard. Theeconomy in material, time, and labor implicit in the practice of themethod is apparent.

It should be understood that changes and modifications in the methodsabove set forth and in the various steps of their production may be madeand substituted for those herein shown and described Without departingfrom the nature and principle of the present invention.

Having thus described my invention, what I claim and desire to secure byLetters Patent is l. The method of making protein standardized reagentsfor use in colorimetry and in checking the accuracy ofspectrophotometric calibration tables as utilized for the determinationof total serum protein, which method oomprises determining the proteincontent in a predetermined quantity of non-human blood plasma, dilutingthe predetermined quantity of plasma in a .85 percent sodium chloridesolution to provide a liquid reagent of predetermined concentration ofprotein, placing a measured quantity of the liquid reagent within aglass container, freezing the liquid material in the container,subjecting the frozen material to a high vacuum system for completedesiccation of the reagent to provide a solid standardized reagent, thensealing the container under vacuum, opening the container at the time ofpreparation of the standard,

' then transferring the reagent into a container of predetermined volumehaving a volume indicating mark, and then making up to the mark withdiluent to constitute a standardized reagent of predeterminedconcentration.

2. The method of making protein standardized reagents for use incolorimetry and in checking the accuracy of spectrophotometriccalibration tables as utilized for the determination of total serumprotein, which method comprises determining the protein content in apredetermined quantity of non-human blood plasma, diluting thepredetermined quantity of plasma in a physiological sodium chloridesolution to provide a liquid reagent of predetermined concentration ofprotein, placing a measured quantity of the liquid reagent within aglass container, freezing the liquid material in the container,subjecting the frozen material to a high vacuum system for completedesiccation of the reagent to provide a solid standardized reagent, thensealing the container under vacuum, opening the container at the time ofpreparation of the standard, then transferring the reagent into acontainer of predetermined volume having a volume indicating mark, andthen making up to the mark with urea thymol solu tion to constitute astandardized reagent of predetermined concentration.

3. The method of making protein standardized reagents for use incolorimetry and in checking the accuracy of spectrophotometriccalibration tables as utilized for the determination of total serumprotein, which method comprises determining the protein content in apredetermined quantity of non-human blood plasma, diluting thepredetermined quantity of plasma in a physiological sodium chloridesolution to provide a liquid reagent of predetermined concentration ofprotein, placing a measured quantity of the liquid reagent within aglass container, freezing the liquid material in the container,subjecting the frozen material to a high vacuum system for completedesiccation of the reagent to provide a solid standardized reagent, thensealing the container under vacuum, opening the container at the time ofpreparation of the standard, flushing the container portions with smallamounts of urea thymol solution until the reagent is dissolved, thentransferring the dissolved reagent into a container of predeterminedvolume having a volume indicating mark, and then making up to the markwith urea thymol solution to constitute a standardized reagent ofpredetermined concentration.

4. The method of making protein standardized reagents for use incolorimetry and in checking the accuracy of spectrophotometriccalibration tables as utilized for the determination of total serumprotein, which method comprises determining the protein content in apredetermined quantity of non-human blood plasma, diluting thepredetermined quantity of plasma in a .85 percent sodium chloridesolution to provide a liquid reagent of predeter mined concentration ofprotein, placing a measured quantity of the liquid reagent within aglass container, freezing the liquid material in the container,subjecting the frozen material to a high vacuum system for completedesiccation of the reagent to provide a solid standardized reagent, thensealing under vacuum the container, opening the container at the time ofpreparation of the standard, flushing the container portions with smallamounts of urea thymol solution until the reagent is dissolved, thentransferring the dissolved reagent into a container of predeterminedvolume, and then making up to the mark with 30 percent urea thymolsolution to constitute a standardized reagent of predeterminedconcentration.

Reichel June 29, 1937 Flosdorf Dec. 24, 1940

1. THE METHOD OF MAKING PROTEIN STANDARDIZED REAGENTS FOR USE INCOLORIMETRY AND IN CHECKING THE ACCURACY OF SPECTROPHOTOMETRICCALIBRATION TABLES AS UTILIZED FOR THE DETERMINATION OF TOTAL SERUMPROTEIN, WHICH METHOD COMPRISES DETERMINING THE PROTEIN CONTENT IN APREDETERMINED QUANTITY OF NON-HUMAN BLOOD PLASMA, DILUTING THEPREDETERMINED QUANTITY OF PLASMA IN A .85 PERCENT SODIUM CHLORIDESOLUTION TO PROVIDE A LIQUID REAGENT OF PREDETERMINED CONCENTRATION OFPROTEIN, PLACING A MEASURED QUANTITY OF THE LIQUID REAGENT WITHIN AGLASS CONTAINER, FREEZING THE LIQUID MATERIAL IN THE CONTAINER,SUBJECTING THE FROZEN MATERIAL TO A HIGH VACUUM SYSTEM FOR COMPLETEDESICCATION OF THE REAGENT TO PROVIDE A SOLID STANDARDIZED REAGENT, THENSEALING THE CONTAINER UNDER VACUUM, OPENING THE CONTAINER AT THE TIME OFPREPARATION OF THE STANDARD, THEN TRANSFERRING THE REAGENT INTO ACONTAINER OF PREDETERMINED VOLUME HAVING A VOLUME INDICATING MARK, ANDTHEN MAKING UP TO THE MARK WITH DILUTENT TO CONSTITUTE A STANDARDIZEDREAGENT OF PREDETERMINED CONCENTRATION.